QIAGEN News Issue No. 1, February 2002 · 2009-05-08 · tative, real-time PCR and RT-PCR using sequence-specific probes — the QuantiTect™ Probe PCR Kit (for PCR and two-step

Innovation Working for YouQIAGEN

FlexiGene technology — for efficient isolation of DNA

QuantiTect™ Probe PCR and RT-PCR Kits — minimize your PCR optimization for

highly sensitive results!

The FlexiGene DNA Kit provides a rapid andconvenient method for purification of DNAfrom variable volumes of human wholeblood, buffy coat, or cultured cells. The simple procedure requires less than 1 hour of hands-on time for purification of DNA from12 samples.

Advantages of the FlexiGene DNA Kitinclude:

◆ Easy handling — purification in a singletube reduces waste and minimizes therisk of sample mix-up

◆ Flexibility in amount of starting material— purification of DNA from 0.1–20 mlwhole blood

◆ Direct purification from whole blood

◆ Speed — protocol involves only 3 shortcentrifugation steps and 3 buffers

◆ No organic extraction

◆ High yields of pure DNA, suitable fordownstream PCR analysis

New

New

QIAGEN introduces two new kits for quanti-tative, real-time PCR and RT-PCR usingsequence-specific probes — the QuantiTect™Probe PCR Kit (for PCR and two-step RT-PCR)and the QuantiTect Probe RT-PCR Kit (for one-step RT-PCR). Both kits are designed for usewith all types of probes, including TaqMan®

or QIAGEN® Operon® dual-labeled probes;LightCycler® hybridization probes or fluores-cence resonance energy transfer (FRET) probes;and Molecular Beacons. The optimized system is designed for use on any real-timePCR cycler, such as the ABI™ (ABI PRISM®

7700 and GeneAmp® 5700), LightCycler(Roche), iCycler™ (Bio-Rad), and DNA EngineOpticon™ (MJ Research) systems.

QuantiTect Probe Kits offer:

◆ Simple assay development — no need for optimization

◆ High sensitivity —stringent built-in hotstart with HotStarTaq™ DNA Polymerase

◆ Easy handling — ready-to-use master-mixformat

◆ Versatility — optimized for use with all types of real-time PCR cyclers andsequence-specific probes

New QuantiTect Probe Kits, page 10

FlexiGene DNA Kit, page 24

1February

2002

2Issue No. 1, 2002

QIAGEN

www.qiagen.com

EditorDouglas J. McGarvey, Ph.D.

Assistant editorsKate E. Bendall, Ph.D., Natalie G. Exton, B.Sc. (Hons.)

Editorial assistantJoanne R. Bailey, B.Sc. (Hons.), M.Sc.

WritersKate E. Bendall, Ph.D., Emma Duncan, Ph.D., Douglas J. McGarvey, Ph.D., Kevin J. Mobbs, Ph.D., Jason Smith, Ph.D., Emma Smythe, Ph.D.

Art directionPetra Kellermann

Graphics and layoutDirk Wirth

Production managementRoland Stelzer

ProductionKatja Zündorf

[email protected]

What’s New?◆ The new QIAGEN® Operon® Product Guide 2002 is included with this issue of QIAGEN News.

QIAGEN Operon (formerly Operon) is a leading supplier of synthetic DNA, providing customoligonucleotides, Array-Ready Oligo Sets™, and custom gene synthesis to life scienceresearchers worldwide.

◆ Did you know that German, French, or Japanese versionsare available of many of our handbooks? Some of these can be downloaded from our web site at www.qiagen.com/literature/handbooks/. Others are available on request; please contact QIAGEN Technical Services for further information.

◆ Don’t miss the new QIAamp® UltraSens™ Virus Kit, described on page 29 of this issue! This kituses new UltraSens technology to concentrate viral RNA and DNA from plasma and serum samples without ultracentrifugation. The resulting highly pure nucleic acids are suitable for usein a wide range of downstream applications.

◆ Are you looking for more sensitivity in ELISA procedures? A new Ni-NTA coating procedureused in the manufacture of Ni-NTA HisSorb™ Strips and Plates significantly increases sensitivityand signal-to-noise ratios, allowing you to use less protein per assay. Find out more on page 18.

◆ We welcome your feedback on QIAGEN News. Please send your comments or suggestions [email protected].

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QIAGENCustomer application article

www.qiagen.com Issue No. 1, 2002

High sensitivity in quantitative, competitiveRT-PCR using QIAGEN® enzymes*

Frédéric Bonino, Julie Milanini, Jacques Pouysségur, and Gilles Pagès

Centre Antoine Lacassagne, Nice, France

This article describes a highly sensitive competitive RT-PCR assay to accurately quantify mRNAlevels of a tumor marker. Use of Omniscript™ RT and the QIAGEN® Taq PCR Master Mix Kit provided high sensitivity and specificity for accurate quantification of low levels of a specificmRNA using small amounts of total RNA.

Vascular endothelial growth factor (VEGF) isimplicated in the progression of a number ofhuman cancers. Its expression correspondswell with tumor growth and metastasis. Highlevels of VEGF correlate with the aggressive-ness of the tumor and poor prognosis (2).

In this paper, we describe a new, competitiveRT-PCR assay based on internal RNA standards for accurate quantification of VEGF mRNA (see box for details about quantification of mRNA using competitiveRT-PCR). Use of Omniscript RT and the TaqPCR Master Mix Kit provided a highly sensitive and specific assay, capable of measuring low levels of VEGF mRNA in smallamounts of total RNA.

Materials and methods

Two oligonucleotides were synthesized, a53mer and 70mer with a 25-base overlap.

The oligo sequences corresponded to bases434 to 559 of the mouse VEGF 164 AmRNA, with a 26-base deletion of bases 457to 482. The oligonucleotides were annealedand the non-annealed regions were filled inusing QIAGEN Taq DNA Polymerase. Theresulting 97 bp DNA fragment was clonedinto a plasmid containing a T7 promoter, andRNA was transcribed from the plasmid invitro using T7 RNA polymerase (Figure 1).

For RNA quantification, differing amounts ofthe in vitro transcribed RNA (1 pg to 10 ng)were added to a set amount of sample RNA(0.5 µg) as an internal competitive standard.Competitive, quantitative RT-PCR was thencarried out using forward and reverseprimers corresponding to sequences identicalin the in vitro transcribed standard RNA andthe sample RNA. These 23mer primers corresponded to the 5' and 3' ends of the

* Excerpted from Bonino, F., Milanini, J., Pouysségur, J., and Pagès, G. (2001) RT-PCR method to quantify vascular endothelial growth factor expression. BioTechniques 30, 1254 (reference 1) with permission from BioTechniques.

Competitive RT-PCR using internal standards

Competitive RT-PCR uses internal standards that are nearly identical to the target sequence andhave the same primer binding sites. The standards are modified slightly from the targetsequence so that amplification products can be differentiated, for example, by restrictiondigestion, gel electrophoresis, or HPLC. RT-PCR is then carried out using known amounts of thestandard. The standards and the target compete for primers to amplify the sequence. Whenthe amounts of standard and target RNA are equal, signals derived from the amplification ofeach are also equal. This allows precise quantification of the target. For competitive RT-PCR,it is important to measure values in the linear amplification phase. As the amplification ratereaches a plateau, quantification becomes less precise.

For absolute quantification of RNA, the copy number or concentration of the nucleic acids used as standards must be known. It is best to use RNA rather than DNA as standards for quantitative RT-PCR since this takes into account variability in the RT reaction dueto differences in RNA quality, sequence, and structure.

competitive RNA construct (bases 559–537and 434–456 of the mouse cDNA). Thus RT-PCR of the sample RNA and the standardRNA produces PCR products with lengths of123 bp and 97 bp, respectively (Figure 1).

For competitive RT-PCR, the reverse-transcriptionstep was carried out using the Omniscript RTKit, priming with the gene-specific reverseprimer described above. PCR was then performed using both gene-specific primers,Taq PCR Master Mix Kit (QIAGEN), and 33P-labeled dATP in a T3 Thermocycler (Biometra) with the following cycling conditions: 95°C for 3 minutes; 28 cycles of95°C for 30 seconds, 56.5°C for 30 seconds, 72°C for 30 seconds; and afinal extension at 72°C for 7 minutes. ThePCR products were resolved by polyacrylamidegel electrophoresis (PAGE) and quantified byphosphorimager analysis on a Fujix Bas1000 Model IPR 1000 (Fuji Photo Film). Thegel clearly resolved the 123 bp fragment,from the sample RNA, and the 97 bp fragment, from the internal standard RNAcontaining the 26 nt deletion.

Results and discussion

We developed a competitive, quantitative RT-PCR assay for quantification of VEGF RNA(Figure 1) as described in “Materials andmethods”. Varying amounts of competitivestandard RNA were added to a set amount ofsample RNA. Since the standard RNAs contained a 26 nt deletion, the sample andcompetitive RNAs were easily separated byPAGE (Figure 2). The relative amounts ofeach RNA were measured using phosphorimager analysis. When the standard and target RNA amounts are equal,signals derived from each of them will also beequal. This amount can be determined graphically by plotting the standard RNAamounts against the ratio of the 2 RNAspecies on a logarithmic scale. The line crossesthe abscissa when the ratio is 1:1 (log 1 = 0),giving the amount of sample RNA (see Figure3A). This method allowed quantification of aslittle as 10–30 pg VEGF RNA, quantities thatare undetectable by northern blot analysis (3).

To validate our RT-PCR method, we looked atthe effect of estradiol stimulation on VEGFmRNA expression in nontransformed CCL39

Issue No. 1, 2002 4

QIAGEN Quantitative, competitive RT-PCR

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Construction of Internal Standards for Competitive RT-PCR

Figure 2 Autoradiogram of a representative set of competitive RT-PCR assays for quantification of VEGFmRNA. Known amounts of competitive RNA, as indicated, were added to 0.5 µg of sample RNA as internalstandards. Two-step RT-PCR was carried out using Omniscript RT and the Taq PCR Master Mix Kit withprimers specific for a 123 nt fragment of the VEGF mRNA. The competitive standard RNA contains a 26 ntdeletion, resulting in a 97 bp amplification product using the same primers. When the standard and targetRNA numbers are equal, signals derived from amplification of each are also equal. C: control with competitive standard RNA alone.

Figure 1 A vector for competitive RNA for internal standards was constructed as described in “Materialsand methods”. The internal standard contains a 26 nt deletion, allowing competitor and sample RNAs to bedistinguished by PAGE.

VEGF mRNA(3' end; 123 bases)

Sample RNA

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Competitive RT-PCR Using Omniscript RT and Taq PCR Master Mix Kits

Competitive RT-PCR

Issue No. 1, 20025

QIAGENQuantitative, competitive RT-PCR

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References

1. Bonino, F., Milanini,J.,Pouysségur, J., andPagès, G. (2001) RT-PCRmethod to quantify vascular endothelialgrowth factor expression. BioTechniques 30, 1254.

2. Lee, Y.H. et al. (1999)Cell-retained isoforms ofvascular endothelialgrowth factor (VEGF) arecorrelated with poor prognosis in osteosarcoma. Eur. J. Cancer 35, 1089.

3. Milanini, J., Vinals, F.,Pouysségur, J., and Pagès,G. (1998) p42/p44 MAPkinase module plays a keyrole in the transcriptionalregulation of vascularendothelial growth factorgene in fibroblasts. J. Biol.Chem. 273, 18,165.

Related articles in this issue

QuantiTect™ Probe PCR andRT-PCR Kits — minimize yourPCR optimization for highlysensitive results! (page 1).

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Chinese hamster lung fibroblasts stablyexpressing a Raf-1:ER chimeric protein. In thissystem, estradiol activates the Raf-1 signal-transduction domain of the chimera via theestrogen receptor (ER). VEGF mRNA is, inturn, induced by the Raf-1 domain, with maximal induction at 5 hours (3).

Using the competitive RT-PCR assay, VEGFmRNA levels were measured in quiescentand estradiol-activated CCL39 cells. Bothbasal and induced VEGF levels were quantifiable, showing that estradiol treatmentcaused a 10-fold induction of VEGF (Figure3A). In contrast, northern blot analysis wasnot sensitive enough to measure basal levels,making it impossible to determine the magnitude of VEGF induction (Figure 3B).

As a further test of the assay, expression ofVEGF was quantified in different cell lineswith varying tumorigenic potential. Highlytransformed cell lines, such as rat colon carcinoma PRO cells, human colon carcinoma HT-29 cells, and Ras-transformedCCL39 cells, express VEGF at 15- to 30-fold

higher levels than quiescent cells (Figure 4).These results correlate with the capacity ofthese cells to induce highly vascular tumors.

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Figure 3 Comparison of sensitivity using competitive, quantitative RT-PCR or northern blot analysis. Total RNA from quiescent CCL39 Raf-1:ER cells (0) and from cells stimulated with estradiol for 5 hours (5 h) was used as sample RNA inboth analyses. ■A Competitive RT-PCR was carried out using Omniscript RT and the Taq PCR Master Mix Kit as describedin “Materials and methods” with 0.5 µg RNA per reaction. The amount of internal standard RNA in each reaction wasplotted against the ratio of the amplification products on a log scale. The amount of VEGF mRNA in the sample is determined from the value where the line crosses the abscissa (arrows). ■B Northern blot analysis using 20 µg total RNAper lane. The arrows indicated 3 differentially spliced forms of VEGF mRNA (all of which are amplified in the RT-PCRassay). The lower panel shows ethidium bromide staining of the 18S rRNA as a control.

VEGF mRNA in Different Cell Types

Figure 4 VEGF mRNA levels were quantified in variouscell types by competitive, quantitative RT-PCR usingOmniscript RT and the Taq PCR Master Mix Kit asdescribed in “Materials and methods”. CCL39: quiescentCCL39 Raf-1:ER cells; Raf: CCL39 Raf-1:ER cells stimulatedfor 5 hours with estradiol; PRO: exponentially growing rat colon carcinoma PRO cells; HT-29: human colon carcinoma HT-29 cells; Ras: Ras-transformed CCL39 cells.

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Highly Sensitive Quantification by Competitive RT-PCR

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Quantitative, competitive RT-PCR


Conclusions

◆ A highly sensitive, competitive, quantitativeRT-PCR assay was developed usingOmniscript RT and the QIAGEN Taq PCRMaster Mix Kit. Using this assay, as littleas 10–30 pg VEGF mRNA could beaccurately quantified. This allows quantification of basal levels that wereimpossible to measure using northern blot analysis.

◆ Using this assay, VEGF mRNA levels couldbe measured using as little as 3 µg totalRNA, comparable to amounts obtainedfrom biopsy samples.

In contrast, a classical ELISA method to evaluate VEGF levels in human serum can only detect significant increases when the tumor has already reached a critical size.

◆ The assay provides a practical tool forresearchers and clinicians to evaluate thepotential of tumor cells to induce highlyaggressive vascularized tumors. The high sensitivity and specificity of Omniscript RTand the Taq PCR Master Mix Kit improvesthe sensitivity and usefulness of the assay,allowing analysis of small amounts of sample. ■

Ordering Information

Product Contents Cat. No.

Omniscript RT Kit — for highly sensitive and specific reverse transcription using 50 ng – 2 µg RNA

Omniscript RT Kit (10)* For 10 reverse-transcription reactions: 20511040 units Omniscript Reverse Transcriptase,10x Buffer RT, dNTP Mix,† RNase-free water

Taq DNA Polymerase — for standard PCR

Taq PCR Master Mix Kit (250 U)* 3 x 1.7 ml Taq PCR Master Mix‡ 201443containing 250 units Taq DNA Polymerase total,3 x 1.7 ml distilled water

Taq DNA Polymerase (250 U)* 250 units Taq DNA Polymerase, 10x PCR Buffer,§ 2012035x Q-Solution, 25 mM MgCl2

Related products

QuantiTect RT-PCR Kits — for quantitative, real-time RT-PCR

QuantiTect SYBR® Green For 200 x 50 µl reactions: 3 x 1.7 ml QuantiTect 204243RT-PCR Kit (200) SYBR Green RT-PCR Master Mix;¶ 1 x 100 µl

QuantiTect RT Mix; 2 x 2.0 ml RNase-free water

QuantiTect Probe RT-PCR Kit (200) For 200 x 50 µl reactions: 3 x 1.7 ml QuantiTect 204443Probe RT-PCR Master Mix;** 1 x 100 µl QuantiTectRT Mix; 2 x 2.0 ml RNase-free water

Oligonucleotide Synthesis Service — high-quality oligos, modified oligos, and longmers

Oligonucleotide Custom-made oligonucleotides up to 100 nt and a InquireSynthesis Service wide range of modified oligos, including Molecular

Beacons, dual-labeled probes, and many more

* Larger kit sizes available: please inquire. § Contains 15 mM MgCl2† Contains 5 mM each dNTP ¶ Contains 5 mM MgCl2

‡ Provides a final concentration of 1.5 mM MgCl2 and 200 µM each ** Contains 8 mM MgCl2

Reader Inquiry No. 02103

Amplification of PCR products longer than3–4 kb is often compromised by nonspecificprimer annealing, suboptimal cycling conditions, and secondary structures in theDNA template. Lengthy optimization is often necessary, by varying factors such as cyclingconditions, primer and dNTP concentrations,and special additives. Here, we describe astandardized, simple protocol for robustamplification of PCR fragments longer than10 kb by the combined use of QIAGEN® Taqand ProofStart™ DNA Polymerases, corre-sponding PCR buffers, and Q-Solution.


Human genomic DNA was isolated fromblood using the QIAamp® DNA Blood MiniKit. PCR was performed using QIAGEN TaqDNA Polymerase, PCR Buffer, Q-Solution,and ProofStart DNA Polymerase. Altern-atively, reactions were carried out using twospecialized long-range PCR kits from Suppliers R and AII, following suppliers’ protocols. One-tenth of each reaction wasloaded onto a 0.8% TAE agarose gel.Cycling programs and reaction mixtures aredetailed in Table 1 (page 9).

Effect of cycling conditions

While depurination is usually not a problemin standard PCR, it can significantly influencethe amplification of longer PCR fragments.This is because longer templates are propor-tionally more depurinated than shorter ones.For this reason, very short denaturation stepsof only 10 seconds give higher yields and nobackground smearing compared to denatu-ration steps of 30 seconds or 1 minute (whichleads to PCR failure; Figure 1A). Extensivedepurination is also observed during the finalextension step. Therefore, using a lowerextension temperature of 68°C instead of72°C dramatically improves yield of longeramplification products (Figure 1B).

Effect of Q-Solution

Secondary structures such as hairpin loops,which are often caused by GC-rich templatestretches, interfere with efficient amplificationof long PCR products. This problem can beovercome by adding reagents that modify themelting behavior of DNA to help resolve secondary structures at lower temperatures.Figure 2 shows that the specificity of amplification of a 4.8 kb PCR fragment was dramatically improved by the addition of Q-Solution.

Issue No. 1, 20027

QIAGEN

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QIAGEN

A new protocol for highly efficient amplification of long PCR products

Susan Kobsch, Katja Decker, and Dirk Löffert

QIAGEN GmbH, Hilden, Germany

M – – + +

– 4.8 kb

– 7.3 kb

Figure 2 A 4.8 kb product from the humantissue plasminogen activator gene was ampli-fied using QIAGEN Taq DNA Polymerase andQIAGEN PCR Buffer in the presence (+) orabsence (–) of Q-Solution. The specific productwas amplified only when Q-Solution wasincluded in the reaction mixture. M: markers.Results from duplicate PCR amplifications areshown.

Effect of Q-Solution

M 1 2 3 M 72°C 68°C

A BEffect of Cycling Conditions

Figure 1 Cycling conditions were as listed inTable 2. ■A To illustrate the effects of prolonged heating of the DNA template or theDNA polymerase, three different PCR cyclingprograms were used to amplify a 7.3 kb fragment of the human interleukin 9 receptorgene. 1: Each PCR cycle had a 60-seconddenaturation step at 94°C. 2: A reaction mix-ture containing Taq DNA Polymerase but lacking the template DNA was incubated at94°C for 30 minutes; the template DNA wasthen added and the PCR cycling program started,using 10-second 94°C denaturation steps.3: The PCR cycling program was comparableto 2, except that there was no additional priorincubation. ■B The 7.3 kb PCR product couldonly be amplified when the temperature of theextension step was lowered from 72°C to68°C. M: markers. Results from duplicate PCRamplifications are shown.

Issue No. 1, 2002 8

QIAGEN Amplification of long PCR products

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Effect of 3'➞ 5' exonuclease activity

Taq DNA Polymerase introduces more errorsinto the PCR product while copying the template than do so-called proofreadingDNA polymerases. Once a mismatch occursduring synthesis, Taq DNA polymerase willeither extend the mismatched strand or fall offthe template strand, leading to mutated orincomplete PCR products, respectively.Although this does not generally affect PCRefficiency when amplifying shorter PCR fragments, amplification of longer PCRproducts can be significantly impaired by mismatches introduced during DNA synthesis.

Proofreading DNA polymerases, such asProofStart DNA Polymerase, usually containan inherent 3'➞ 5' exonuclease activity thatremoves base-pair mismatches. Adding asmall amount of ProofStart DNA Polymeraseto the PCR mixture therefore significantlyimproves the amplification efficiency oflonger PCR products. This is shown by comparing PCR mixtures containing, in addition to Taq DNA Polymerase, eitherProofStart DNA Polymerase or a proofreadingpolymerase with point mutations to inactivatethe 3'➞ 5' exonuclease activity (Supplier SIII).PCR mixtures containing ProofStart DNA Polymerase consistently enable amplification

of long products with high yield (Figure 3).These results emphasize the importance of the3'➞ 5' exonuclease activity to correct misincorporated bases, increasing PCRefficiency, especially for longer PCR products.

Combined effect of 3'➞ 5' exonuclease, hotstart, PCR Buffer, and Q-Solution

The parameters described above are summarized in a final, optimized protocol (Tables 1 and 2) using QIAGEN Taq DNA Polymerase, PCR Buffer, Q-Solution, andProofStart DNA Polymerase. This optimized protocol was then compared with commercially available kits for long-rangePCR. Two different template–primer systemswere tested, generating 9.3 kb and 15 kbPCR products. The optimized mixture ofQIAGEN DNA polymerases and reagentsgave superior amplification yields of the specific PCR product in all cases (Figure 4).This is due to the reduction of secondary structures in the template by Q-Solution andthe efficient removal of misincorporatedbases by ProofStart DNA Polymerase. Speci-ficity of the reaction was further increased bythe inactive state of ProofStart DNA Polymerase before the initial activation step(hot start). The lack of ProofStart exonucleaseactivity before this hot start prevents primerdegradation during reaction setup and theinitial heating phase of the thermal cycler.

Conclusions

◆ The new protocol presented here enablesamplification of long PCR products withhigh yields and specificity, without theneed for time-consuming optimization.

◆ The simple hot start provided by ProofStart DNA Polymerase eliminates theneed for manual hot-start procedures ortwo-mix setup strategies that are oftenrecommended in other protocols.

◆ The efficiency, specificity, and ease ofhandling offered by QIAGEN DNA polymerases, buffers, and reagents leadsto genuine time savings in reaction setupand amplification procedures.

Figure 3 A 12.3 kb fragment of the human interleukin 9 receptor gene was amplified fromhuman genomic DNA using QIAGEN Taq DNAPolymerase, either with an exonuclease-deficient proofreading enzyme mutant from Supplier SIII (–)or with ProofStart DNA Polymerase (+) containingthe exonuclease activity. Results from duplicatePCR amplifications are shown. M: markers.

Effect of 3'➞ 5' Exonuclease Activity

M – +

– 12.3 kb

A B

Optimized Reaction Conditions for Longer PCR Products

QIAGEN

QIAGEN

M R AII

– 9.3 kb– 15 kb

Figure 4 Long PCR products were amplified from human genomic DNA using either the optimized QIAGENreagent mixture (QIAGEN), a commercially available mixture for long-range PCR from Supplier R (R) or a commercially available mixture for long-range PCR from Supplier AII (AII). Amplification was performed according to suppliers’ protocols. Equal volumes were analyzed. Primers were designed to amplify ■A a 9.3 kb fragment of the human tissue plasminogen activator gene or ■B a 15 kb fragment of the human coagulation factor IX gene. M: markers.

M R AII

Issue No. 1, 20029

QIAGENAmplification of long PCR products

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Table 1. PCR components for long amplification products (master mix and template)

Component Volume/reaction Final concentration

PCR Buffer,* 10x 5 µl 1x(supplied with QIAGEN Taq DNA Polymerase)

Q-Solution, 5x 10 µl 1x

dNTP mix (10 mM of each) 1.5 µl 300 µM of each dNTP

Primer A variable 0.5 µM

Primer B variable 0.5 µM

Taq DNA Polymerase 1 µl 5 units

ProofStart DNA Polymerase (diluted)† 1 µl 0.2 units

Distilled water variable

Template DNA variable 200 ng – 1 µg for genomic DNA or5–50 ng for plasmidor λ DNA

Total volume 50 µl

* Contains 15 mM MgCl2† Dilute enzyme in 1x ProofStart PCR Buffer to a concentration of 0.2 units/µl.

Table 2. Cycling conditions for amplifying longer PCR products

Initial activation step 2 min 95°C

3-step cycling:Denaturation 10 s 94°C

Annealing 1 min 50–68°C ‡

Extension 1 min/kb 68°C

Number of cycles 35–40

End of PCR cycling Indefinite 4°C

‡ Approximately 5°C below Tm of primers



Taq DNA Polymerase (250 U) 250 units Taq DNA Polymerase, 20120310x PCR Buffer,§ 5x Q-Solution,25 mM MgCl2

Taq PCR Core Kit (250 U) 250 units Taq DNA Polymerase, 20122310x PCR Buffer,§ 5x Q-Solution, 25 mM MgCl2, dNTP Mix¶

ProofStart DNA Polymerase (100 U) 100 units ProofStart DNA Polymerase, 20220310x ProofStart PCR Buffer,** 5x Q-Solution, 25 mM MgSO4

§ Contains 15 mM MgCl2¶ Contains 10 mM each dNTP ** Contains 15 mM MgSO4


■

Issue No. 1, 2002 10

QIAGEN

continued from page 1

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New QuantiTect Probe Kits

Simple assay development with minimaloptimization

Developing quantitative, real-time PCR andRT-PCR assays with fluorescent probes oftenrequires extensive and time-consuming optimization procedures. This can involvecomplex, simultaneous adjustment of primer,probe, and magnesium concentrations in acoordinated matrix. QIAGEN QuantiTectProbe Kits minimize or even eliminate theneed for optimization, making assay development both simple and fast.

Optimal results can be achieved the first time,using the recommended, pre-optimized primerand probe concentrations given in the QuantiTect Probe protocols. This greatly minimizes the need to adjust reaction parameters.

A balanced combination of KCl and(NH4)2SO4 in the specially formulated buffer provides a greater tolerance to variable magnesium concentrations. The Mg2+ concen-tration in the buffer is already optimized so that,in most cases, the user does not need to experiment with different magnesium concen-trations for each new target (Figure 1).

High sensitivity for reliable quantification

PCR sensitivity is directly related to the speci-ficity of the assay. Extension of nonspecificallyannealed primers and primer-dimers can occurat low temperatures during reaction setup andthe first denaturation step. A particular prob-lem with low-copy targets is the increasedopportunities for these artifacts due to the highratio of primers and probes to the smallamount of target sequences. High backgrounddue to nonspecific products can block out specific signals, meaning that accurate detec-tion is only possible after more cycles.

HotStarTaq DNA Polymerase, in both kits,provides a hot start to the PCR for highly specific and sensitive amplification. The hotstart prevents nonspecific products, primer-dimer formation, and background,and increases the yields of specific PCR products. This leads to increased sensitivity, allowing detection of low-copy targets thatcould otherwise be obscured by high back-ground levels.

In addition to HotStarTaq DNA Polymerase,the QuantiTect Probe RT-PCR Kit contains aunique blend of Sensiscript™ and Omniscript™

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Supplier R,optimized Mg2+ concentrationB CA

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Figure 1 Real-time PCR analysis of a fragment of the human protease inhibitor 1 gene was carried out using FRET probes and the QuantiTect Probe PCR Kit or a kit from Supplier R. Analyses were performed on the LightCycler system. Reactions contained human genomic DNA with the indicated copy numbers. ■A QIAGEN, with Mg2+ concen-tration supplied ■B Supplier R, with Mg2+ concentration supplied ■C Supplier R, with optimized Mg2+ concentration (2.5 mM). NTC: no template control.

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QIAGEN

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New QuantiTect Probe Kits

Reverse Transcriptases. The combination ofboth enzymes ensures highly efficient and sensitive reverse transcription over a widerange of template amounts (1 pg to 500 ng)without optimization. Both reverse transcriptaseshave high affinities for RNA, providing efficientsynthesis of full-length cDNAs, even throughregions with complex secondary structure.

Compared with other real-time RT-PCR kitsthat use sequence-specific probes, the QuantiTect Probe RT-PCR Kit provides significantly higher sensitivity for accuratereal-time quantification (Figure 2).

Easy handling with convenient master-mixformat

Both QuantiTect Probe Kits contain a uniquemaster mix, with HotStarTaq DNA Polymerase, an optimized PCR or RT-PCRbuffer, and dNTPs. dUTP is included,enabling an optional UNG (uracil-N-glycosy-

lase) treatment. In addition, both mastermixes contain the internal reference dye ROX.This allows normalization on ABI sequencedetection systems without interfering withreactions on other real-time cyclers that do nothave this normalization option. Master mixesfor both kits are stable at –20°C and 4°C formany months. Storage at 4°C allows evenfaster setup of amplification reactions by eliminating thawing time.

The QuantiTect Probe RT-PCR Kit also containsa blend of Omniscript and SensiscriptReverse Transcriptases, provided as a separate solution to enable simple setup ofcontrol reactions without reverse transcriptases.RNase-free water for the addition of primersand templates is provided with both kits. Thisready-to-use format minimizes pipetting stepsand eliminates tedious calculations, maximizing both convenience and accuracy(see flowchart).

Figure 2 Amplification plots of real-time RT-PCR analysis using the ABI PRISM 7700 Sequence Detection System (Applied Biosystems). One-step, quantitative RT-PCR was carried out using the QuantiTect Probe RT-PCR Kit (QIAGEN) or one-step, quantitative RT-PCR kits from suppliers AII, L, or SIII, as indicated, according to suppliers’ instructions. Reactions were performed with the indicated number of copies of an in vitro transcript of the TATA-box binding protein (TBP). Inset: Agarose-gel analyses of end-point PCR results. M: markers.

PCR and two-step RT-PCR

Add primersand templates

Distribute

Quantitativereal-time PCR

or

PCR Master Mix

M

QIAGEN

Supplier L Supplier SIII

105 104 103 102 0

105

104

103

102

0

105

104

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104

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M 105 104 103 102 0

Highly Sensitive Quantification Using the QuantiTect Probe RT-PCR Kit

Issue No. 1, 2002 12

QIAGEN

www.qiagen.com

QIAGEN New QuantiTect Probe Kits

Related articles in this issue

High sensitivity in quantita-tive, competitive RT-PCR usingQIAGEN enzymes, page 3.

www.qiagen.com



QuantiTect Probe Kits — for quantitative, real-time PCR and RT-PCR using sequence-specific probes

QuantiTect Probe PCR Kit (200) For 200 x 50 µl reactions: 3 x 1.7 ml QuantiTect 204343Probe PCR Master Mix;* 2 x 2.0 ml RNase-free water

QuantiTect Probe RT-PCR Kit (200) For 200 x 50 µl reactions: 3 x 1.7 ml QuantiTect 204443Probe RT-PCR Master Mix;* 1 x 100 µl QuantiTect RT Mix; 2 x 2.0 ml RNase-free water

* Contains 8 mM MgCl2

For related products, see page 6.


Versatile use with any real-time cycler andfluorescent probe

QuantiTect Probe Kits can be used with alltypes of fluorescent probes for real-time PCRand RT-PCR. Compatible probes include TaqMan or other dual-labeled probes thatrely on the 5'➞ 3' exonuclease activity of TaqDNA polymerase, as well as LightCyclerhybridization probes or FRET probes, and

Molecular Beacons. Both kits containdetailed, optimized protocols for using theseprobes with the ABI, LightCycler, iCycler, orDNA Engine Opticon systems (Table 1).

QIAGEN Operon offers a wide variety ofthese and other modified custom oligos.Please contact QIAGEN Technical Servicesfor more details. ■

Table 1. Optimized protocols for QuantiTect Probe Kits

Detection system

Probes

ABI Sequence DNA EngineDetection LightCycler iCycler Opticon Systems (Roche) (Bio-Rad) (MJ Research)

TaqMan or dual-labeled probes ✔ ✔ ✔ ✔

Molecular Beacons ✔ ✔ ✔ ✔

LightCycler hybridization n.t. ✔ n.t. n.t.or FRET probes

✔ : Optimal for use with QuantiTect Probe Kitsn.t.: not tested

13

QIAGEN


Following the article “Important consider-ations for generating transgenic mice”(QIAGEN News 2001 No. 5), this articlediscusses important factors when generatingknockout mice, as well as an overview of theprocedure.

Gene targeting technology uses homologousrecombination to replace a specific region ofthe genome with a different DNA sequence.In knockout animals, a specific gene isreplaced by vector sequences so that thegene no longer functions. Such animals areused in a variety of ways and allow theresearcher to test the specific function of agene by monitoring the effect of absence ofits protein product. “Knock-in” animals canalso be generated where the sequence of agiven gene is changed. Mice are most commonly used in these studies since they aresmall and easy to handle, reproduce quickly,and have genes that are easy to manipulate.Mice provide valuable models of human disease since the genetic patterns of miceand humans are similar.

In contrast to the knockout technique, whichalters the gene(s) of a host cell, generatingtransgenic mice involves adding genes to thechromosomes within the egg and can involvethe transfer of genes from one species toanother. Knockout mice take around 14months to engineer, which is considerablylonger than transgenic mice, which takearound 6 months. This is because geneknockout technology is much more specific.

Generation of knockout mice

The process of creating a knockout mouse follows several basic steps (see flowchart). Firstly, a gene-targeting construct must be engineered, and then transfected into murineembryonic stem (ES) cells, usually via electroporation. ES cells are pluripotent andcan be maintained indefinitely in an undifferentiated state. However, under the correct conditions ES cells are capable of differentiating into any cell type.

Important considerations for generating knockout mice

ES cells

x

x

Prepare targeting vector

Transfect ES cells

Analyze DNA samples

Mouse homozygousfor gene knockout

Procedure for GeneratingKnockout Mice

QIAGEN Plasmid KitsQIAquick Gel Extraction Kits/QIAEX II Gel Extraction Kits

Analyze DNA samples

Breed heterozygous mice

Breed chimeric mouse

Confirm homologous recombinationby PCR or Southern blotting

Select for transfectants

DNeasy Tissue KitsHotStarTaq DNA Polymerase

Transfer blastocyst topseudo-pregnant female

Microinject transfected ES cellsinto blastocyst



Issue No. 1, 2002 14

QIAGEN Considerations for generating knockout mice

www.qiagen.com

Transfected ES cells are selected, and homologous recombinants are identifiedusing PCR and Southern blotting. Successfullytransfected stem cells are then microinjectedinto a blastocyst, and blastocysts are transferred to a pseudo-pregnant female. A proportion of the offspring have some tissuesderived from the original blastocysts (with awild-type genotype), and others derived fromthe manipulated ES cells (with the alteredgenotype). ES cell lines and donor blastocysts from mice with different naturalcoat colors are used so that in the chimericmouse, skin cells derived from the ES cells willbe one color and cells derived from the blastocyst will be another. These offspring arecalled chimeric, and are easily identified byhaving 2 coat colors.

Chimeric mice are then back- and self-crossedover several generations in order to generatehomozygous knockout mice. The time takenfor this process can be substantially shortenedby using marker-assisted techniques, alsoknown as “speed congenics”. Using this technology the number of generations necessary to develop a congenic strain isconsiderably reduced (1, 2).

Important considerations

Vector preparation

The design of the vector is perhaps the mostimportant step in the generation of knockoutmice. The composition of the vector has amajor impact on transfection efficiency andhence the ability to select and screen ES celltransformants, and also on whether the genein question is actually knocked out.

A typical targeting vector comprises a plasmid DNA backbone, along with selectionmarkers and DNA homologous to the regionsflanking the gene to be knocked out. The plasmid vector usually contains an origin ofreplication and a selective cassette, which contains antibiotic selection markers that areused to discriminate between transfected anduntransfected ES cells. The plasmid backboneprovides the framework for DNA manipulation within the host. QIAGEN®

EndoFree® Plasmid Kits can be used to isolateultrapure, endotoxin-free plasmid DNA whichis highly suited for vector preparation.

QIAquick® and QIAEX® II Gel Extraction Kitscan be used for purification of linear DNAfragments from agarose gels following restriction digestion reactions.

Another important factor in designing the target vector is the DNA flanking the selectivecassette of the plasmid DNA. This flankingDNA should be derived from the genome ofthe ES cell line into which the targeting construct will be transfected, and homologousto the regions surrounding the gene to beknocked out. The longer the homologousregions, the greater the targeting efficiency.As a general guide it is recommended toinclude 3–8 kb of homologous DNA on eitherside of the selective cassette.

Screening for homologous recombinantclones

Once the vector has been transfected into EScells and transfected clones have been isolated, clones that have undergone homologous recombination for the gene ofinterest must be selected, and differentiatedfrom those that have undergone random (non-homologous) recombination. This isdone by PCR or Southern blotting.

PCR is usually used as a preliminary methodof screening, and Southern blotting is used toconfirm the identity of positive clones.DNeasy® Tissue Kits are highly suited for isolation of genomic DNA, yielding DNA thatperforms well in both PCR reactions andSouthern blotting analyses (3, 4). HotStarTaq™

DNA Polymerase is well suited for highly specific PCR.

Screening mice for gene knockout

Once chimeric mice are obtained they arebred with wild-type mice to produce heterozygous offspring. These heterozygousprogeny are then back- and self-crossed anumber of times in order to generate pupshomozygous for the knockout gene. At allstages, progeny are screened for the knockout gene using Southern blotting or PCRanalysis. DNeasy Tissue and DNeasy 96 Tissue Kits have been successfully used for isolation of DNA from mouse tails whenscreening knockout mice (3, 4, 5, 6).

References

1. Markel, P. et al. (1997)Theoretical and empiricalissues for marker-assistedbreeding of congenicmouse strains. NatureGenet. 17, 280.

2. Wakeland, E., Morel, L.,Achey, K., Yui, M.,Longmate, J. (1997)

Speed congenics: a classical technique inthe fast lane (relativelyspeaking). Immunol.Today 18, 472.

3. Levy, J., Montross, L.,Andrews, N. (2000)Genes that modify thehemochromatosis phenotype in mice. J.Clin. Invest. 105, 1209.

4. Saari, J. et al. (2001)Visual cycle impairment incellular retinaldehydebinding protein (CRALBP)knockout mice results indelayed dark adaptation.Neuron 29, 739.

5. Doyonas, R. et al. (2001)Anuria, omphalocele, andperinatal lethality in micelacking the CD34-relatedprotein podocalyxin. J.Exp. Med. 194, 13.

6. Iakoubova, O. et al.(2000) Microsatellite marker panels for use inhigh-throughput genotypingof mouse crosses. Physiol.Genomics 3, 145.

Issue No. 1, 200215

QIAGENConsiderations for generating knockout mice

www.qiagen.com

Summary

◆ Vector design is the most critical step inthe generation of knockout mice.QIAGEN Plasmid Kits and DNA CleanupSystems ensure high-quality DNA for usein vector construction.

◆ Accurate identification of recombinantclones and pups homozygous for geneknockout is crucial. DNeasy Tissue Kitsprovide a rapid and reliable method forisolating DNA from ES cells and mousetails, while HotStarTaq DNA Polymerasefacilitates high-specificity PCR. ■



EndoFree Plasmid Kits — for purification of endotoxin-free ultrapure plasmid DNA

EndoFree Plasmid Maxi Kit (10)* 10 QIAGEN-tip 500, 10 QIAfilter™ 12362Maxi Cartridges, Buffers

QIAquick Gel Extraction Kits — for purification of 70 bp – 10 kb DNA fragments

QIAquick Gel Extraction Kit (50)† 50 QIAquick Spin Columns 28704

QIAEX II Gel Extraction Kits — for purification of 40 bp – 50 kb DNA fragments

QIAEX II Gel Extraction Kit (150)† For up to 150 extractions: QIAEX II 20021Suspension, Buffers

HotStarTaq DNA Polymerase — for highly specific, robust amplification

HotStarTaq DNA Polymerase (250)† 250 units HotStarTaq DNA Polymerase, 20320310x PCR Buffer‡, 5x Q-solution, 25 mM MgCl2

DNeasy Tissue Kits — for isolation of genomic DNA from animal tissues and cells, yeast, or bacteria

DNeasy Tissue Kit (50)† 50 DNeasy Spin Columns, Reagents and Buffers, 69504Collection Tubes (2 ml)

DNeasy 96 Tissue Kits§ — for high-throughput isolation of DNA from animal tissues and cells

DNeasy 96 Tissue Kit (4)† For 4 x 96 DNA minipreps: 4 x DNeasy 96 Plates, 69581Reagents and Buffers, Collection Microtubes (1.2 ml), Collection Microtube Caps, 96-Well Plate Registers

*Different kit formats available; please inquire† Larger kit sizes available; please inquire‡ Contains 15 mM MgCl2§ Requires the use of the QIAGEN 96-Well-Plate Centrifuge System

As a leading supplier of knockout andtransgenic mice and related services,efficient and reliable mouse genotypingtools are of paramount importance. TheDNeasy 96 Tissue Kit and QIAGENHotStarTaq DNA Polymerase consis-tently meet our high standards, andprovide excellent results.

Jan L. SeymourSupervisor, Molecular AnalysisTaconic Biotechnologywww.taconic.com

“

”


Issue No. 1, 2002 16

QIAGEN

www.qiagen.com

Efficient cDNA labeling and cleanup forsuccessful microarray analysis

Christian Korfhage, Ivonne Schröder-Stumberger, and Evelyn Fisch

QIAGEN GmbH, Hilden, Germany

Tissue or cultured cells

RNA

Labeled cDNA

Hybridization on chips

Array constructed with

PCR fragments

RNA purification using theRNeasy System

cDNA labeling using Omniscript RT

cDNA cleanup using the QIAquick System and extra GuHCI wash

PCR fragment cleanup usingthe QIAquick 96 System

Production of Target DNA The rapidly expanding field of microarrayanalysis has led to many important advancesin gene expression profiling and mutationanalysis. Improvements in array manufacturenow allow simultaneous quantitative analysisof RNA transcription levels across an entiregenome. As the technique has developed,microarray technology has become considerably more robust and sensitive. However, one of the most important factorsfor successful analysis remains the quality ofthe nucleic acids used in the procedure.

For reliable results, microarrays must beconstructed using reproducible amounts ofprobe DNA (the DNA that is spotted ontochips), and the labeled target DNA used forhybridization must be of a high quality andpurity.

Problems mainly occur during the productionof target DNA. Nonspecific priming duringthe labeling step can misrepresent the geneexpression profile by producing extraneouscDNAs from rRNA, multiple cDNAs from different regions of the same mRNA, or shortcDNAs that hybridize nonspecifically (1). Inaddition, carryover of salts, unincorporatedlabeled nucleotides, and proteins during thesubsequent cleanup step can further compro-mise the purity of target DNA. The presenceof any one of these contaminants in the targetDNA often leads to high background andartifactual signals.

We describe here a procedure using Omniscript™ Reverse Transcriptase for cyanine-5 (Cy®5) labeling of target DNA followed by cleanup using a modifiedQIAquick® PCR Purification Kit protocol (see flowchart). The QIAquick cleanup proce-dure delivered high yields of high-purity target cDNA that gave strong signal intensi-ties and minimal background in microarrayanalysis.


Total RNA was purified from mouse brainusing the RNeasy® Mini Kit. Labeled targetcDNA was generated by reverse transcription using Omniscript RT in a reaction containing Cy5-dCTP and 10 µgtotal RNA in a total volume of 50 µl. Cleanupof target cDNA was carried out using a modified QIAquick PCR Purification Kit protocol containing an additional wash step.This wash step, using 35% (w/v) guanidinehydrochloride, is carried out after cDNAbinding and ensures maximum removal ofunincorporated nucleotides. Hybridizationwas performed using target cDNA corresponding to 2 µg total RNA.

Semiquantitative PCR was used to estimatethe yield of cDNA recovered after theQIAquick cleanup procedure. 1/2500th ofthe labeling reaction or the eluate followingcleanup was added to a PCR containing aprimer that amplified a 199 bp fragment.Half of each PCR was loaded onto anagarose gel and quantified by densitometry.

Labeled cDNA Unlabeled cDNA

High Recoveries of Labeled cDNA

M b a b a M

Figure 1 Semiquantitative PCR analysis of cDNAbefore (b) and after (a) the modified QIAquick procedure. Calculated average recoveries were 89%(labeled cDNA) and 91% (unlabeled cDNA) of input.M: markers.

Issue No. 1, 200217

QIAGEN

www.qiagen.com

Results

Figure 1 shows agarose gel analysis of thePCR products obtained in the semiquantitativePCR analysis. On average, 89% of thelabeled input cDNA was recovered.

Figure 2 shows the results of two independentmicroarray analyses using target cDNAcleaned up using the modified QIAquick protocol. The absence of significant back-ground and high reproducibility in thehybridized arrays demonstrates the superiorresults obtainable with QIAquick purified target cDNA.

Conclusions

Generation of target cDNA using Omniscript RT for the reverse transcriptionlabeling reaction, followed by DNA cleanupusing a modified QIAquick PCR purificationprotocol, gives high signal intensities with minimal background and reliable, reproducible results in microarray analysis. ■

References

1. Fisch, E., Brinker-Krieger,N., Schwarz, H., Schäfer,A., and Korfhage, C.(2001) Improved signal-to-noise ratios using Omniscript RTfor microarray and LightCycler® analyses.QIAGEN News 2001 No. 3, 13.

Efficient cDNA labeling and cleanup for successful microarray analysis

Figure 2 Two independent microarray analyses using the same QIAquick 96 purified probe DNA derived from mouse brain. Cy5-labeled target cDNA wascleaned up using a modified QIAquick procedure as detailed in “Materials andmethods”.

Reproducible Microarray Analysis with High Signal Intensitiesand Low Background




QIAquick 96 PCR Purification Kit (4)* For purification of 1 x 96 PCR reactions: 281811 QIAquick 96 Plate, Buffers, Collection Microtubes (1.2 ml), Caps

QIAquick PCR Purification Kit (50)* For purification of 50 PCR reactions: 2810450 QIAquick Spin Columns, Buffers, Collection Tubes (2 ml)

Omniscript RT Kit (50)* For 10 reverse-transcription reactions: 20511140 units Omniscript Reverse Transcriptase, 10x Buffer RT, dNTP Mix,t RNase-free water

RNeasy Mini Kit (50)* 50 RNeasy Mini Spin Columns, 74104Collection Tubes (1.5 ml and 2 ml), RNase-free Reagents and Buffers

* Other kit sizes available; please inquire.t Contains 15 mM each dNTP

Issue No. 1, 2002 18

QIAGEN


Increased sensitivity in bioassays usingimproved Ni-NTA HisSorb™ Plates and Strips

Ni-NTA HisSorb™ Plates and Strips allow efficient, directed binding of 6xHis-taggedproteins — even from crude lysates — andsubsequent assay using standard ELISA techniques and equipment. A new Ni-NTAcoating procedure used in the manufacture ofHisSorb Strips and Plates delivers significantly increased sensitivity in ELISA procedures.

The Ni-NTA–6xHis-tag interaction

Ni-NTA HisSorb Plates and Strips utilize theremarkable selectivity of Ni-NTA (nickel-nitrilotriacetic acid) for an affinity tag of sixconsecutive histidine residues (the 6xHis tag).Proteins, immobilized via an N- or C-terminal6xHis tag, are optimally presented to anti-bodies or other interaction partners in a con-venient and reproducible procedure.

The directed binding enhances assay sensitivity,minimizes nonspecific binding, and increasesreproducibility and signal-to-noise ratios. Byusing the Ni-NTA–6xHis tag interaction forimmobilization, time-consuming protein-specific optimization trials are eliminated.Now, due to a new coating procedure usedin their manufacture, assays using HisSorbPlates and Strips are even more sensitive.

Increased sensitivity in assays

The improved HisSorb Strips and Plates offerenhanced 6xHis-tag binding efficiency andminimized nonspecific binding, significantlyincreasing the sensitivity of assays comparedto former HisSorb products and other commercially available plates (Figures 1 and 2).This increase in sensitivity means that lessmaterial is required for each assay, which isespecially important when assaying preciousor rare samples.

Wide choice of formats

Ni-NTA HisSorb Strips are supplied framedand sealed in sets of twelve 8-well strips formaximum convenience when testing or opti-mizing assays using a limited number of samples. For higher throughput requirements,

0.5

1.0

1.5

2.0

2.5

3.0

50 100 150 200 250 300

6xHis-tagged TNFα (ng/ml)

New HisSorb PlateFormer HisSorb PlateSupplier P

Improved Sensitivity of New HisSorb Plates

AU

00

1.0

2.0

3.0

4.0

5.0

6xHis-tagged Thioredoxin-Tag·100 (ng/ml)

New HisSorb PlateFormer HisSorb Plate

AU

Assay Using Tag·100 Antibody

00 1 2 3 4 5 6

Figure 1 Dilutions (150 µl) of 6xHis-tagged tumor necrosis factor α (TNFα) were bound to a new HisSorb Plate(◆ ), a former HisSorb Plate (■ ), or a metal chelate-coated plate from Supplier P (▲) for 30 minutes at room temperature, and washed 4 times. A peroxidase-conjugated anti-TNFα antibody (150 µl at 50 mU/ml) wasadded and allowed to bind for 30 minutes at room temperature. After four washes, color was developed using 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS).

Figure 2 Dilutions (200 µl) of thioredoxin tagged with 6xHis at the N-terminus and with Tag·100 at the C-terminus were bound to a new HisSorb Plate (◆ ) and a former HisSorb Plate (■ ) for 60 minutes at room temperature. The plate wells were washed four times and 200 µl of Tag·100 Antibody was added and allowedto bind for 60 minutes at room temperature. After 4 washes, 200 µl of a peroxidase-conjugated anti-mouse IgGsecondary antibody was added and allowed to bind for 60 minutes at room temperature. After an additional 4washes, color was developed using ABTS.

New

Ni-NTA HisSorb plates in 96- and 384-well formats

Issue No. 1, 200219

QIAGEN

www.qiagen.com

Improved sensitivity in assays with Ni-NTA HisSorb Plates and Strips

96-well Ni-NTA HisSorb Plates are available.QIAGEN also offers a wide range of customized assay products to suit your individual requirements.* Ni-NTA HisSorbPlates in 96- and 384-well formats are available in transparent format for use withcolorimetric assays and a white or blackopaque format for luminescence- or fluorescence-based assays. All plates arecompatible with standard multichannel pipetsand automated plate washers and readers.

Reliable and reproducible assay results

All HisSorb products are charged with nickeland preblocked with BSA for immediate use.Vigorous quality control measures ensure thatHisSorb products provide uniform binding,washing, and optical properties for reliableand reproducible measurements, both on awell-to-well and a plate-to-plate basis.

The Tag·100 Antibody

The Tag·100 Antibody is used to detect proteins,expressed using the pQE-100 DoubleTag™

vector, that carry a 6xHis tag at their N-terminus and the Tag·100 epitope at theirC-terminus (Figure 3). Directed immobiliza-tion using the Ni-NTA–6xHis tag interaction,

and detection of the optimally presentedTag·100 epitope using the Tag·100 Antibody,increases standardization in ELISA procedures.

The high levels of sensitivity, convenience,and flexibility offered by HisSorb Plates andStrips make them the method of choice forassay of 6xHis-tagged proteins. To find outmore about the QIAexpress® Assay Systemvisit us at www.qiagen.com or contactQIAGEN Technical Services or your local distributor. ■



Ni-NTA HisSorb Strips (24) 2 racks of 12 x Ni-NTA–coated 8-well strips 35023in 96-well format

Ni-NTA HisSorb Plates (5) 5 Ni-NTA–coated, transparent 96-well plates 35061

Ni-NTA HisSorb Plates, white (5) 5 Ni-NTA-coated, opaque, white 96-well plates 35081

Related products

Tag·100 Antibody, BSA-free (100 µg) 100 µg mouse anti-Tag·100 (lyophilized, 34680BSA-free, for 1000–2500 ml working solution for ELISA)

pQE-100 DoubleTag Vector DNA 25 µg pQE-100 (lyophilized) 33003

* Please inquire about customized assay products available in minimum orders of 100 plates (allow 6 weeks for delivery). Customized products available include Ni-NTA HisSorb Plates (transparent, white, or black) in 96- and 384-well format, as well as plates coated with any of the QIAexpress Anti·His Antibodies in 96- or 384-well format.


Tag·100 Antibody

Protein of choice

6xHis tag

Ni-NTA

Tag·100

Figure 3 Detection of proteinsexpressed by the pQE-100 Double-Tag vector using Tag·100 Antibodies.

Tag·100 Antibodies

Issue No. 1, 2002 20

QIAGEN L A B A U T O M A T I O N

Contact your QIAGEN Sales Representative or visit us at www.qiageninstruments.comto find out how you can automate your life science applications today!

BioRobot® Twister™ robotic arms are versatile systems for the transfer and storage ofmicroplates and other labware. Twister robotic arms are used with BioRobot 3000and BioRobot 8000 workstations to allow integration of external instruments forwalkaway automation of sequential tasks. BioRobot Twister I and Twister II roboticarms are compatible with most laboratory instruments, ensuring seamless integrationinto your workflow.

Using BioRobot Twister robotic arms with BioRobot workstations allows integration ofinstruments such as thermal cyclers, spectrophotometers, plate sealers, and storageincubators. Walkaway automation of sequential tasks such as sample purification,normalization of sample concentration, reaction setup and cycling, and reactioncleanup is now simple.

BioRobot Twister robotic arms provide:

◆ More instrument integration options

◆ Fast transfer of microplates

◆ Unattended overnight processing capability

◆ On-board storage for up to 320 microplates

Contact QIAGEN today to discover improved system integration!

BioRobot® Twister™ robotic arms — expand your integration options

New


21

QIAGEN


The QIAamp® UltraSens™ Virus Kit uses newUltraSens technology for increased viralnucleic acid yield from plasma and serumsamples. Now your downstream assays canbe even more sensitive!

In the QIAamp UltraSens Virus procedure,UltraSens technology is first used to captureviral nucleic acids from a 1 ml sample, allowing them to be highly concentrated. No ultracentrifugation or specialized laboratoryequipment is required, and an internal controlcan be added at the start of the

procedure. The concentrated nucleic acidsare then purified using a fast silica-gel–basedspin-column procedure, yielding highly pure,ready-to-use nucleic acids suitable for use inamplification-based assays.

The QIAamp UltraSens Virus Kit is suitable forpurifying nucleic acids from viruses such asHIV-1, HAV, HBV, HCV, and B19V (see Table 1),as well as BHV-1, SIV, PCV, and PCMV. It isalso well suited for efficient isolation ofgenomic DNA from serum and plasma. ■

UltraSens™ technology for highly efficientpurification of viral nucleic acids

Table 1. Highly sensitive detection of viruses using UltraSens technology

Virus Detection limit Detection method*

HCV 1.8 IU/ml AMPLICOR®

HIV 10 IU/ml AMPLICOR

HBV 15 genome equivalents/ml TaqMan®

HAV 4 genome equivalents/ml TaqMan

B19V 16 genome equivalents/ml TaqMan

* Viral nucleic acids were amplified and detected using either the Cobas® AMPLICOR or TaqMan systems from Roche Molecular Systems, Inc.

New



QIAamp UltraSens Virus Kit (50) 50 viral nucleic acid preps: 50 QIAamp 53704Spin Columns, QIAGEN Proteinase K, Carrier RNA, Collection Tubes (2 ml), Buffers

QIAamp UltraSens Virus Kit (250) 250 viral nucleic acid preps: 250 QIAamp 53706Spin Columns, QIAGEN Proteinase K, Carrier RNA, Collection Tubes (2 ml), Buffers


Issue No. 1, 2002 22

QIAGEN Customer application article

www.qiagen.com

Anthrax is a potentially fatal infectious disease caused by a spore-forming bacterium,Bacillus anthracis. Approximately 95% ofanthrax cases in humans result from exposureto infected soil or animals, through eitheringestion or skin lesions. Rapid diagnosis isimportant for treating the disease, as antiobiotics are highly effective if adminis-tered to an infected individual before symptoms occur. A reliable and fast methodfor detection of B. anthracis in soil samples istherefore important for disease control. PCR-based assays should provide more rapidmethods for detection of anthrax than protocols requiring bacterial cultivation.

An important factor in the success of PCR isthe use of high-quality DNA templates. Preparation of high-quality DNA from soilsamples is challenging for two reasons. Firstly, soil contains high levels of enzymaticinhibitors that will interfere with downstreamPCR, and secondly, samples are often largein volume. Furthermore, DNA purity, andhence PCR detection limits, depend on soiltype. For example, sandy soils yield the highest quality DNA, while it is very difficultto isolate amplifiable DNA from rich loamysoils containing high levels of humic acid.This is because the loam binds DNA efficiently,and humic acid is a potent PCR inhibitor.

We describe here the development of a rapidmethod for the isolation of B. anthracis DNAfrom loamy soil samples. Soil samples wereinoculated with inactivated B. anthracis cellsand DNA was isolated using the QIAampDNA Stool Mini Kit according to a modified

protocol. The resulting DNA was of a highquality and performed well in downstreamPCR.


Soil samples (5 g samples of sandy loam)were inoculated with 1 ml solutions containingeither 105 or 107 inactivated B. anthraciscells. B. anthracis cells were inactivated usingH2O2, resulting in bacterial inactivation without cell lysis. Samples were left for 5 minutes at room temperature, then 1 mlwater was added to each tube to form soilsuspensions. Samples were incubated in awater bath at 100°C for 10 minutes, and centrifuged for 5 minutes at 3000 rpm. Thesupernatant was removed and 7 volumes ofBuffer ASL was added.

For DNA isolation, 2 InhibitEX™ tablets fromthe QIAamp DNA Stool Mini Kit were addedto each tube, tubes were incubated for 1 minute, and the contents were mixed. Tubeswere centrifuged for 4 minutes at 10,000 x gand supernatants were transferred to newtubes. One volume of Buffer AL was added toeach sample, tubes were mixed by inversion,and one volume of 96–100% ethanol wasadded.

Washing and elution were carried out usingthe QIAvac 24. Samples were applied tocolumns from the QIAamp DNA Blood MidiKit and washed with 1 ml volumes of BuffersAW1 and AW2. The columns were transferred to 15 ml tubes and centrifuged for15 minutes at 4500 rpm. For elution, 300 µlwater was pipetted onto each column,

Rapid isolation of anthrax DNAfrom large-volume soil samples using

QIAamp® KitsGudrun Zoll, Gudrun Grote, Roland Dierstein, and Stefan Köhne

Armed Forces Scientific Institute for Protection Technologies, Munster, Germany

QIAamp® Kits were used to develop a rapid method for the isolation of Bacillus anthracis DNAfrom large-volume soil samples. The method provided good yields of high-quality DNA thatperformed well in downstream PCR.

Issue No. 1, 200223

QIAGENRapid isolation of anthrax DNA

www.qiagen.com

columns were incubated for 5 minutes atroom temperature, and centrifuged for 5 minutes at 4500 rpm. The eluate containingDNA was applied again to the column and asecond elution step was performed, usingidentical conditions.

The resulting DNA was used as a template inPCR using QIAGEN® Taq DNA Polymerase.Each reaction contained 2 µl DNA templateand 16 pmol each primer. Soil supernatantsspiked with B. anthracis, and DNA purifiedfrom these supernatants were both used astemplates in quantitative, real-time PCR usingthe LightCycler®. Each reaction contained 2 µl DNA template, 16 pmol primer, andQIAGEN Taq DNA Polymerase.

Results and discussion

Successful detection of B. anthracis DNA fromsoil samples was achieved using quantitative,real-time PCR with the LightCycler (see Figure 1).Figure 2 shows the improved PCR sensitivityachieved by removing the inhibitors presentin loam. Using supernatants from spiked soilas PCR templates, B. anthracis could bedetected in samples inoculated with 107

B. anthracis cells/5 g soil. Following DNApurification, this sensitivity improved so that105 B. anthracis cells /5 g soil were detected.The same sensitivity was found in positivecontrol samples. The purification proceduretherefore efficiently removed the many PCRinhibitors present in loamy soil.

Conclusions

QIAamp Kits were used to develop a methodfor the rapid isolation of bacterial DNA fromlarge volumes of soil. This method reduces thetime required for DNA isolation by 1 hour incomparison with previously published methods.The QIAamp method provided efficientremoval of PCR inhibitors, and B. anthraciscould be detected down to a sensitivity of 105 cells/5g soil. ■


Figure 2 Real-time PCR products: negative controls (lanes 1, 4, and 7); products from soilsupernatants spiked with 105 (lane 2) and 107

(lane 3) B. anthracis cells/5 g soil; products fromDNA purified from soil supernatants spiked with105 (lane 5) and 107 (lane 6) B. anthraciscells/5 g soil; positive controls (lanes 8 and 9);markers (M).

Figure 1 Quantitative, real-time PCR using theLightCycler. Samples 1–9 are the products shownin Figure 2, lanes 1–9.

Real-Time PCR Analysis

High Sensitivity Following DNA PurificationM 1 2 3 4 5 6 7 8 9 M

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QIAamp DNA Stool Mini Kit (50) For 50 DNA preps: 50 QIAamp Spin 51504Columns, QIAGEN Proteinase K,InhibitEX tablets, Buffers, Collection Tubes (2 ml)

QIAamp DNA Blood Midi Kit (20)* For 20 DNA midipreps: 20 QIAamp Midi 51183Spin Columns, QIAGEN Protease, Buffers, Collection Tubes (15 ml)

QIAvac 24 Vacuum manifold for processing 1–24 spin 19403columns: includes QIAvac 24 Base, Lid, Luer Caps

* Larger kit sizes avaiable; please inquire

Issue No. 1, 2002 24

QIAGEN

www.qiagen.com

FlexiGene DNA Kit

The FlexiGene protocol begins with additionof lysis buffer to samples. Cell nuclei and mitochondria are pelleted by centrifugationand resuspended in denaturation buffer containing QIAGEN Protease. Following protein digestion, DNA is precipitated by addition of isopropanol, recovered by

centrifugation, washed in 70% ethanol, anddried. DNA is resuspended in hydrationbuffer and is ready for direct use in down-stream assays or storage at –20°C. The efficient performance of the DNA in PCRassays is shown in Figure 1. ■

Efficient Downstream PCR Analysis

M M M

Hugl1 ng template

tRNAlys/ATPase10 pg template

M M M

Hugl10 ng template

tRNAlys/ATPase10 ng template

M M M

Hugl1 µg template

tRNAlys/ATPase1 µg template



FlexiGene DNA Kit (50) For purification of DNA from 50 ml whole blood: 51204Buffers, QIAGEN Protease

FlexiGene DNA Kit (250) For purification of DNA from 250 ml whole blood: 51206Buffers, QIAGEN Protease

Figure 1 Variable amounts of DNA templatewere used to amplify the single-copy Hugl geneand a mitochondrial target (tRNAlys/ATPase).Each sample was analyzed 6 times, and repro-ducible results were achieved. M: markers.

continued from page 1


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Practical

Hints

The QIAGEN Guide to AnimalCell Culture

Part II: Safety and handling considerations for animal cell culture

Welcome to the second of a new series of articles aimed at providing useful hints for culturing animal cells. This article describes considerations for working with animal cell cultures as well asinformation on cell culture contamination. The series will continue in future issues of QIAGEN Newswith information on cell culture growth conditions, followed by cell culture protocols.

Legislation and regulatory guidelines

Before undertaking any work with human or animal tissue (e.g., to establish a primary cell culture), it is necessary to ensure that the nature of the work conforms to the appropriate medical-ethical and animal-experiment legislation and guidelines. It may be necessary to seekapproval from the relevant regulatory authorities and/or individuals.

Safety considerations and biohazards

When working with potentially hazardous material, it is important to be aware of the possiblerisks associated with both the material and the experimental protocol. All cell cultures are considered a biohazard because of their potential to harbor an infectious agent (e.g., a virus).The degree of hazard depends on the cells being used and the experimental protocol. Primarycell cultures in particular should be handled carefully as these cultures have a high risk of containing undetected viruses. Although commonly used cell lines are generally assumed to befree of infectious agents, care should still be exercised when working with these cell lines as itis possible that they contain infectious agents, such as latent viruses. Cell cultures used to studyspecific viruses should be assumed to have the same degree of hazard as the virus under study.

We recommend handling all material as potentially infectious to ensure the safest possible working environment. Work should be performed in an approved laminar flow hood usingaseptic technique, and the creation of aerosols should be avoided (see below). After the workis complete, all waste media and equipment (i.e., used flasks, pipets, etc.) should be disinfectedby autoclaving or immersion in a suitable disinfectant according to institutional and regionalguidelines.

Handling cell cultures

Adherence to good laboratory practice when working with cell cultures is essential for two reasons: first, to reduce the risk of exposure of the worker to any potentially infectious

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References

1. Freshney, R.I. (1993) Culture of Animal Cells, a Manual of Basic Technique. 3rd ed. NewYork: Wiley-Liss.

2. Spector, D., Goldman,R.R., and Leinwand, L.A.,eds. (1998) Cells: a Laboratory Manual.Cold Spring Harbor, NY:Cold Spring Harbor Laboratory Press.

3. Drexler, H.G. et al., eds.(1997) DSMZ Catalog ofHuman and Animal CellLines. 6th ed.

26

agent(s) in the cell culture, and second, to prevent contamination of the cell culture with micro-bial or other animal cells (see below).

Aseptic technique and minimization of aerosols

Aseptic technique and the proper use of laboratory equipment are essential when working withcell cultures. Always use sterile equipment and reagents, and wash hands, reagent bottles, andwork surfaces with a biocide or 70% ethanol before beginning work.

Creation of aerosols should be avoided — aerosols represent an inhalation hazard, and canpotentially lead to cross-contamination between cultures. To avoid aerosols, use TD (to deliver)pipets, and not TC (to contain) pipets; use pipets plugged with cotton; do not mix liquids byrapidly pipetting up and down; do not use excessive force to expel material from pipets; anddo not bubble air through liquids with a pipet. Avoid releasing the contents of a pipet from aheight into the receiving vessel. Expel liquids as close as possible to the level of liquid of thereceiving vessel, or allow the liquid to run down the sides of the vessel.

Proper use of equipment can also help minimize the risk of aerosols. For example, when usinga centrifuge, ensure the vessel to be centrifuged is properly sealed, avoid drops of liquid nearthe top of the vessel, and use centrifuge buckets with caps and sealed centrifuge heads to prevent contamination by aerosols.

Laminar flow hoods

For the most efficient operation, laminar flow hoods should be located in an area of the laboratory where there is minimal disturbance to air currents. Avoid placing laminar flow hoodsnear doorways, air vents, or locations where there is high activity. Hoods are often placed indedicated cell culture rooms.

Keep laminar flow hoods clean, and avoid storing equipment inside the hood.

Before starting work, disinfect the work surface of the hood as well as the outside of any bottles (e.g., by wiping with 70% ethanol), and then place everything needed for the cell culture procedure in the hood.

Arrange equipment, pipets, waste containers, and reagent bottles so that used items are not placed near clean items, and avoid passing used items over clean items.

Place used items (e.g., pipets) in a container inside the hood, and disinfect or seal before removing from the hood.

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Contamination

The presence of microorganisms can inhibit cell growth, kill cells, and lead to inconsistentresults. Contamination of cell cultures can occur with both cell culture novices and experts. Potential contamination routes are numerous. For example, cultures can be infected throughpoor handling, from contaminated media, reagents, and equipment (e.g., pipets), and frommicroorganisms present in incubators, refrigerators, and laminar flow hoods, as well as on theskin of the worker and in cultures coming from other laboratories.

Bacteria, yeasts, fungi, molds, mycoplasmas, and other cell cultures are common contaminantsin animal cell culture. To safeguard against accidental cell culture loss by contamination, we recommend freezing aliquots of cultured cells to re-establish the culture if necessary. A protocolfor freezing cell cultures will be provided in a future issue of QIAGEN News.

Microbial contamination

The characteristic features of microbial contamination are presented in Table 1. The presenceof an infectious agent sometimes can be detected by turbidity and a sharp change in the pH ofthe medium (usually indicated by a change in the color of the medium), and/or cell culturedeath. However, for some infections, no turbidity is observed and adverse affects on the cellsare not easily observed.

Cell cultures should be routinely evaluated for contamination. Mycoplasmal infections are oneof the more common and difficult-to-detect infections; their detection and eradication aredescribed in further detail below.

Mycoplasmal infection — detection

Mycoplasmas are small, slow-growing prokaryotes that lack a cell wall and commonly infectcell cultures. They are generally unaffected by the antibiotics commonly used against bacteriaand fungi. Furthermore, as mycoplasma do not overgrow cell cultures and typically do not causeturbidity, they can go undetected for long periods of time and can easily spread to other cellcultures. The negative effects of mycoplasmal contamination include inhibition of metabolismand growth, as well as interference with nucleic acid synthesis and cell antigenicity. Acute infection causes total deterioration of the cell culture, sometimes with a few apparently resistantcolonies that may, in fact, also be chronically infected. There are two main approaches to

Table 1. Characteristic features of microbial contamination

Characteristic Bacteria Yeast Fungi

Change in pH pH drop with most pH change with pH sometimesinfections heavy infections increases

Cloudy medium: Shimmering in spaces Round or ovoid Thin filamentous Under microscope between cells; rods or particles that bud mycelia; sometimes(100–400x) cocci may be observed off smaller particles clumps of spores

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QIAGEN E X T R A S

detect mycoplasma — Hoechst 33258 staining (1, 2) and mycoplasma-specific DNA probes(Fisher Scientific). Alternatively, a PCR-based, mycoplasma-testing service is offered by the ATCC(www.atcc.org) or BioReliance (www.biomeva.com) on a fee-for-service basis.

Mycoplasmal infection — eradication

The best action to take with a culture containing chronic mycoplasmal infection is to discard itby either autoclaving or incineration. Only if the cell culture is absolutely irreplaceable shoulderadication be attempted. This process should be performed by experienced personnel in anisolated hood that is not used for cell culture, preferably in a separate room. Elimination ofmycoplasma is commonly achieved by treatment with various commercially available antibioticssuch as a quinolone derivative (Mycoplasma Removal Agent), ciprofolxacin (Ciprobay®),enrofloxacin (Baytril®), and a combination of tiamulin and minocycline (BM-Cyclin). Treatmentprocedures and appropriate antibiotic concentrations can be found in the suppliers’ instructionsand in references 1 and 3.

Cross-contamination of cell lines

Cross-contamination of one cell culture with fast-growing cells from another culture (such asHeLa) presents a serious risk. To avoid cross-contamination, only use cell lines from a reputablecell bank; only work with one cell line at a time in the hood; use different pipets, bottles ofreagents, and bottles of media for different cell lines; and check cells regularly for the correctmorphological and growth characteristics.

The QIAGEN Guide to Animal Cell Culture will continue in future issues of QIAGEN News withinformation on cell culture growth conditions and cell culture protocols. If there is any other information you would like to see on these pages of QIAGEN News, please let us know bycalling QIAGEN Technical Services or your local distributor. ■

Issue No. 1, 2002 29

QIAGENE X T R A S

www.qiagen.com

QIAGEN Technical Services are always available to answer your questions! QIA-Hints

Please do not hesitate to call your local QIAGEN Technical Service Department if you have any questions or require further information regarding any QIAGENproducts.

DNA

Q Our laboratory has developed itsown set of optimized buffers andconditions for lysing specific

bacteria. I’ve already lysed the bacteriausing our lab’s protocol. Can I still usethe DNeasy® Tissue Kit or the QIAamp®

DNA Mini Kit to isolate genomic DNAfrom them?

A If you have already optimized lysis conditions for a specific sample source and don’twant to alter them, you can still use the DNeasy or QIAamp Kits for isolation of genomic DNA. Simply lyse the sample in your lysis buffer, and follow the protocol for

isolation of genomic DNA from crude lysates. For cultured bacteria, see page 28 of theDNeasy Tissue Kit Handbook (April 1999), or, if you are working with human clinical samples containing bacteria, see page 52 of the QIAamp DNA Mini Kit and QIAamp DNABlood Mini Kit Handbook (September 2001).

TRANSFECTION

Q Can I use PolyFect® TransfectionReagent for other cell lines,besides the 5 that it’s been

optimized for?A PolyFect Transfection Reagent is designed to provide optimal transfection results with

COS-7, NIH/3T3, HeLa, 293, and CHO cells. We expect that other cell lines may alsobe successfully transfected using PolyFect Transfection Reagent, but we do not have

detailed information. Therefore, it may be necessary to optimize transfection conditions foryour specific cell type and DNA vector.

To choose the best reagent, a searchable list of cell lines and primary cells that have beensuccessfully transfected using QIAGEN® Transfection Reagents is available at the QIAGENTransfection Tools web site (www.qiagen.com/transfectiontools/). If your cell type of interest isnot listed, or if you want to test different reagents to optimize transfection, QIAGEN offers theTransfection Reagent Selection Kit. This kit contains both Effectene® and SuperFect®

Transfection Reagents and a comprehensive handbook to guide you through optimization anddetermine the best reagent and transfection conditions.

Issue No. 1, 2002 30

QIAGEN E X T R A S


Come and See UsQIAGEN exhibits and presents at many venues throughout the year. During the next few months, you canvisit us at any of the meetings listed below. Come and see what’s new, or talk to a company representative.

In North America

American Academy of Forensic Science (AAFS) 11–16 February, Atlanta, GA

CHI — Genome Tri-Conference 2002 23 February – 1 March, Santa Clara, CA

Association for Biomolecular Research (ABRF) 9–12 March, San Diego, CAResource Facilities

Pittcon 2002 17–22 March, New Orleans, LA

American Association for Cancer Research (AACR) 6–10 April, San Francisco, CA

Microarrays Through Macroresults 3 19 April – 1 May, Boston, MA

Experimental Biology 2002: Translating the Genome 20–24 April, New Orleans, LA

Clinical Virology Symposium (CVS) 28 April – 1 May, Clearwater Beach, FL

In Europe

Swiss Society for Microbiology (SGM/SSM) 20–21 February, Lucerne, Switzerland

Union of the Swiss Societies of Experimental Biology 7–8 March, Lugano, Switzerland(USGEB/USSBE)

Forum Labo Biotech 26–29 March, Paris, France

DNA Vaccines 2002 10–12 April, Edinburgh, UK

ANALYTICA 23–26 April, Munich, Germany

In Asia and Australasia

27th Annual Conference on Protein Structure 10–14 February, Lorne, Australia and Function

14th Lorne Cancer Conference 14–17 February, Lorne, Australia

23rd Lorne Genome Conference 17–21 February, Lorne, Australia

Issue No. 1, 2002 31

QIAGENE X T R A S

www.qiagen.com

Trademarks and disclaimers

Patented or patent-pending technology and/or registered or registration-pending trademarks of the QIAGEN Group of Companies:QIAGEN®, QIAamp®, QIAEX®, QIAexpress®, QIAquick®, Array-Ready Oligo Sets™, BioRobot®, DNeasy®, Effectene®, EndoFree®,HisSorb™, HotStarTaq™, InhibitEX™, Ni-NTA, Omniscript™, Operon®, PolyFect®, ProofStart™, QuantiTect™, RNeasy®,Sensiscript™, Superfect®, Twister™, UltraSens™.

ABI is a trademark of Applera Corporation or its subsidiaries.

ABI PRISM is a registered trademark of Applera Corporation or its subsidiaries.

AMPLICOR and COBAS are registered trademarks of Roche Molecular Systems, Inc., licensed to Roche Diagnostic Systems, Inc.

Baytril and Ciprobay are registered trademarks of Bayer AG.

Cy is a registered trademark of Amersham Pharmacia Biotech Limited or its subsidiaries.

DNA Engine Opticon is a trademark of MJ Research.

GeneAmp and TaqMan are registered trademarks of Roche Molecular Systems, Inc.

iCycler is a trademark of Bio-Rad Laboratories, Inc.

LightCycler is a registered trademark of Idaho Technology Inc.

SYBR is a registered trademark of Molecular Probes, Inc.

Registered names, trademarks, etc. used in this document, even when not specifically marked as such, are not to be consideredunprotected by law.

QIAGEN Robotic Systems are not available in all countries; please inquire.

QIAGEN sample preparation products may be used in clinical diagnostic laboratory systems after the laboratory has validated theircomplete system as required by CLIA ’88 regulations in the U.S. or equivalents in other countries. QIAGEN products are for researchpurposes only unless cleared for a specific purpose by the appropriate regulatory authorities.

Purchase of QIAGEN products for PCR is accompanied by a limited license to use them in the Polymerase Chain Reaction (PCR)process for research and development activities in conjunction with a thermal cycler whose use in the automated performance of thePCR process is covered by the up-front license fee, either by payment to Perkin-Elmer or as purchased, i.e. an authorized thermalcycler. The PCR process is covered by U.S. Patents 4,683,195 and 4,683,202 and foreign equivalents owned by Hoffmann-LaRoche AG.

Hoffmann-La Roche owns patents and patent applications pertaining to the application of Ni-NTA resin (Patent series: RAN4100/63: USP 4.877.830, USP 5.047.513, EP 253 303 B1), and to 6xHis-coding vectors and His-labeled proteins (Patent series:USP 5.284.933, USP 5.130.663, EP 282 042 B1). All purification of recombinant proteins by Ni-NTA chromatography for commer-cial purposes, and the commercial use of proteins so purified, require a license from Hoffmann-La Roche.

© 2001 QIAGEN, all rights reserved

QIAGEN I N T H I S I S S U E

DNA cleanup

Efficient cDNA labeling and cleanup for successful microarray analysisOmniscript RT and QIAquick® Kits deliver high-quality fluorescently labeled target cDNA for microarray procedures 16

DNA isolation from animal samples

Important considerations for generating knockout miceQIAGEN Kits provide high-quality DNA for generation and screening of knockout mice 13

DNA isolation from clinical samples

FlexiGene technology — for efficient isolation of DNANew technology for DNA purification from clinical samples 1

UltraSens™ technology for highly efficient purification of viral nucleic acidsNew technology for purification of viral RNA and DNA 21

Rapid isolation of anthrax DNA from large-volume soil samples using QIAamp® KitsCustomer article Efficient removal of inhibitors from soil using QIAamp Kits 22

PCR and RT-PCR

QuantiTect™ Probe PCR and RT-PCR Kits — minimize your PCR optimization for highly sensitive results!New kits for quantitative, real-time PCR and RT-PCR using fluorescent probes 1

High sensitivity in quantitative, competitive RT-PCR usingQIAGEN® enzymesCustomer article Omniscript™ RT and the Taq PCR MasterMix Kit for highly sensitive quantification of a tumor marker 3

A new protocol for highly efficient amplification of long PCR productsA combination of ProofStart™ and Taq DNA Polymerases for optimal long-range PCR 7

Protein assay

Increased sensitivity in bioassays using improved Ni-NTA HisSorb™ Plates and StripsNew coating procedure increases sensitivity in assays using 6xHis-tagged proteins 18

QIAGEN Instruments

BioRobot® Twister™ robotic arms — expand your integration optionsRobotic arms allow integration of sequential tasks 20

Extras

Practical Hints — The QIAGEN Guide to Animal Cell Culture 25

QIA-Hints 29

Meetings and exhibitions 30

New

New

New

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Argentina Tecnolab S.A. (011) 4555 0010 Austria/Slovenia Merck Eurolab GmbH (01) 576 00 0 Belgium/Luxemburg Westburg b.v.0800-1-9815 Brazil Uniscience do Brasil 011 3622 2320 China Gene Company Limited (852)2896-6283 Cyprus Scientronics Ltd(02) 765 416 Czech Republic BIO-CONSULT spol. s.r.o. (420) 2 417 29 792 Denmark Merck Eurolab A/S 43 86 87 88 Egypt Clinilab52 57 212 Finland Merck Eurolab Oy (09)-804 551 Greece BioAnalytica S.A. (01)-640 03 18 India Genetix (011)-542 1714or (011)-515 9346 Israel Westburg (Israel) Ltd. 08 6650813/4 or 1-800 20 22 20 Korea LRS Laboratories, Inc. (02) 924-86 97Malaysia RESEARCH BIOLABS SDN. BHD. (603)-8070 3101 Mexico Quimica Valaner S.A. de C.V. (55) 55 25 57 25 The NetherlandsWestburg b.v. (033)-4950094 New Zealand Biolab Scientific Ltd. (09) 980 6700 or 0800 933 966 Norway Merck Eurolab AS22 90 00 00 Poland Syngen Biotech Sp.z.o.o. (071) 351 41 06 or 0601 70 60 07 Portugal IZASA PORTUGAL, LDA (21) 424 7312Singapore Research Biolabs Pte Ltd 2731066 Slovak Republic BIO-CONSULT Slovakia spol. s.r.o. (02) 5022 1336 South Africa SouthernCross Biotechnology (Pty) Ltd (021) 671 5166 Spain IZASA, S.A. (93) 902.20.30.90 Sweden Merck Eurolab AB (08) 621 34 00Taiwan TAIGEN Bioscience Corporation (02) 2880 2913 Thailand Theera Trading Co. Ltd. (02) 412-5672 In other countries contact:QIAGEN, Germany

ItalyTel. 02-33430411 Fax 02-33430426

USATel. 800-426-8157Fax 800-718-2056

CanadaTel. 800-572-9613Fax 800-713-5951

Germany Tel. 02103-29-12400Fax 02103-29-22000

JapanTel. 03-5547-0811Fax 03-5547-0818

SwitzerlandTel. 061-319-30-31Fax 061-319-30-33

UK and IrelandTel. 01293-422-999Fax 01293-422-922

www.qiagen.com

FranceTel. 01-60-920-930Fax 01-60-920-925

QIAGEN: Distributors: AustraliaTel. 03-9489-3666Fax 03-9489-3888

Documents

QIAGEN News Issue No. 1, February 2002 · 2009-05-08 · tative, real-time PCR and RT-PCR using sequence-specific probes — the QuantiTect™ Probe PCR Kit (for PCR and two-step